微能量超音波細胞刺激載台之設計

Abstract

背景介紹: 再生醫學研究日益興起，而如何利用外在的刺激來加速細胞的反應，是再生醫學研究在未來能更蓬勃發展的重點之一。至今有不少的生物學者相信超音波的物理刺激能加速細胞的增生速率或影響細胞內部的蛋白質反應，且提出不少研究成果加以佐證，但其成果較缺少觀察細胞接收超音波刺激的過程變化。然而，超音波細胞刺激過程相關研究雖然早在於2006年已被發表，但至今相關研究所使用之超音波刺激載台都有面臨載台體積限制、顯微鏡透光與空間能量強度不一致之問題。 研究目的: 本論文研究目的為將超音波刺激載台有系統地結合於顯微鏡進行即時觀測與開發出更適用於超音波細胞刺激實驗之載台。本研究共開發出Ludin, LIC, MIGO, Probe Adapter四種刺激載台，以下分別介紹： Ludin Chamber: 本研究最初設計乃將超音波能量導入常應用於顯微鏡觀察之Ludin Chamber中，並結合顯微鏡組成一套超音波刺激細胞即時觀測系統。在細胞實驗設定隨著時間依序為觀察10至20分的控制組、提供1 MHz連續超音波刺激40至60分的超音波刺激組與觀察停止超音波刺激約40分的恢復組，而觀察的細胞分別有A549(非小細胞癌細胞)、EpH4_WT細胞、EpH4_EE2細胞與EpH4_U3細胞。在設備參數檢驗方面為利用水聽計(Hydrophone)量測超音波刺激空間的能量場分布狀況、超音波強度，可發現此超音波刺激系統能提供刺激空間一均勻能量場分布，且量測之最大超音波強度(ISATA)為1.08 mW/cm2。另一方面，本研究亦利用熱電偶量測此系統在超音波導入時，刺激空間的溫度變化狀況為何。從量測結果可發現在恆溫箱溫度為33oC，且同時施打四顆1 MHz超音波晶片時，刺激空間溫度最終穩定於38 oC。

LIC: LIC 為根據Ludin Chamber在實驗應用上所遇到的問題作為設計基礎下所誕生出的產品，此設計同樣能使超音波能量在玻片形成洩漏藍姆波(Leaky Lamb waves)，將能量傳遞至刺激空間之水溶液中。此外，LIC特點為直接將超音波晶片黏固於不鏽鋼底座上表面，故本設備更專屬於超音波刺激細胞之觀測實驗使用。LIC的主要應用同樣為超音波刺激即時觀測實驗，而觀察的細胞為C2C12細胞。另一方面，LIC亦可單純作為超音波刺激細胞之工具提供實驗人員使用，如應用於細胞遷移實驗與EpH4¬_WT細胞貼附觀測。在設備參數檢驗方面為利用水聽計(Hydrophone)量測超音波刺激空間的能量場分布狀況、超音波強度，可發現此超音波刺激系統亦能提供刺激空間一均勻能量場分布，且量測施打兩顆1 MHz超音波晶片之正逆接法超音波強度(ISATA)分別為0.44 mW/cm2與0.17 mW/cm2。同樣地，本研究亦利用熱電偶量測此系統在超音波導入時，刺激空間的溫度變化狀況為何。從量測結果可發現在室溫為26oC，且同時施打四顆1 MHz超音波晶片時，刺激空間溫度最終約穩定於27.8 oC。 MIGO: MIGO為參考LIC配合模式與超音波傳遞模式所設計而成。而本研究進一步與中研院共同合作進行超音波神經細胞離子通道測試實驗，其目的在於探討超音波刺激是否能開啟細胞膜上的離子通道進而影響細胞內部機能。然而，因應用於電生理實驗，故設計上需考量達到低電訊號干擾與刺激空間絕緣之問題。此外，MIGO的另一個設計特點為具較大的內部刺激空間，可直接對培養皿內的細胞進行超音波刺激。在細胞實驗方面除了利用細胞膜片箝制技術(Patch-Clamp Technique )來偵測DRG神經節細胞接受超音波刺激時的離子通道狀況外，亦有用來刺激培養皿中的C2C12細胞以觀察細胞分化狀況。在設備參數檢驗同樣利用水聽計(Hydrophone)量測超音波刺激空間的能量場分布狀況、超音波強度，可發現此超音波刺激載台能提供刺激空間一均勻能量場分布，且量測之最大超音波強度(ISATA)為1.19 mW/cm2，而培養皿中為0.84 mW/cm2。本研究亦利用熱電偶量測此系統在超音波導入時，刺激空間的溫度變化狀況為何。從量測結果可發現在恆溫箱溫度為33oC，且同時施打四顆1 MHz超音波晶片時，刺激空間溫度最終約穩定於36.6 oC。 Probe Adapter: Probe Adapter的設計目的為解決MIGO在超音波神經細胞離子通道測試實驗中所遇到的問題，而開發出的新一套超音波刺激細胞的方式。其設計特色在於提供一連結超音波晶片的光滑貼附面，與一配合前端夾持玻璃圓管針尖之不鏽鋼移液管的內孔。此架設的優點在於可將超音波傳遞至玻璃針尖做局部範圍的輸出。根據應用於超音波神經細胞離子通道測試實驗結果，可發現偵測到細胞膜電流變化的成功率提高，進而提升實驗之可重複性。在設備檢驗方面為利用水聽計(Hydrophone)量測不同版本的Probe Adapter在振幅為2Vpp、3Vpp、5Vpp、8Vpp、10Vpp的1.1 MHz連續超音波輸入時，玻璃針尖所輸出的能量約為0.006至0.02 mW/cm2。 結論： 本論文所介紹的四種超音波刺激設備，除了Ludin Chamber為市售產品而不納入實驗應用統整外，其餘的LIC、MIGO與Probe Adapter皆為自行開發之產品，分別應用於超音波刺激即時觀測實驗、培養皿超音波刺激實驗及超音波神經細胞離子通道測試實驗中，並從實驗結果判斷本開發之設備能達到預期之功能。

Introduction: Regenerative medicine research is on the rise, and how to use external stimulation to accelerate cell response is one of the focuses in the future. As of now, many biologists believe that physical stimulation by ultrasound can accelerate the rate of cell proliferation. Many research results support this belief, but there is no direct observation of cellular response throughout the stimulation process. Although live cell imaging with ultrasound stimulation has been published since 2006, most of the ultrasonic stimulation devices used by relevant research institutes had the limitation of the device size, source of light being blocked on the microscope, and inconsistent spatial energy intensity.

Objective: The objective of this thesis is to combine the device for ultrasound stimulation with microscopes for real-time cell observations, and develop devices that are more suitable for cellular stimulation.

Ludin Chamber: We started by incorporating ultrasonic energy into the Ludin Chamber, which is often used in microscopy, and combined it with a microscope to turn it into an ultrasound-stimulated cellular real-time observation system. In the cell experiment we set the control group as 10 to 20 minutes, and then provide 1 MHz continuous ultrasound stimulation for about 40 to 60 minutes, and finally observe cell recovery for 40 minutes. The cells observed were A549 (NSCLC), EpH4_WT, EpH4_EE2 and EpH4_U3. In the device parameter measurement, the energy field distribution and ultrasonic intensity within the chamber space are measured by hydrophone. The results demonstrate that the ultrasonic stimulation system can provide a uniform energy field in the chamber, and the maximum ultrasonic intensity (ISATA) is 1.08 mW/cm2. On the other hand, this study also measured the temperature change of the stimulation space by thermocouple when the device was turned on. when the temperature of the incubator is 33oC and four 1 MHz ultrasonic probes were applied, the temperature in the chamber was stabilized at 38 oC.

LIC: LIC is an improved device based on the design problems encountered by Ludin in experiments. This design also enables ultrasonic energy to form Leaky Lamb waves on the slides, subsequently transmitting ultrasound energy into the liquid. In addition, LIC has a special design that adhered and fixed ultrasonic probes to the upper surface of stainless steel base, so the device is more specifically used for observation experiments of ultrasonic stimulator cells. The main application of LIC is as same as Ludin. We demonstrated its usage with experiments on C2C12 cells. On the other hand, LIC can also be used as a tool for ultrasound stimulation, such as cell migration experiments and EpH4_WT attachment observations. As device parameters, the results demonstrate that the ultrasonic stimulation system can provide a uniform energy field in the chamber, and the intensity (ISATA) of the positive connection method and the reverse connection method are 0.44 mW/cm2 and 0.17 mW/cm2 respectively. Similarly, the results demonstrated that when the temperature of the ambient is 26oC and four 1 MHz ultrasonic probes were applied, the temperature in the chamber was stabilized at 27.8 oC in the end.

MIGO: MIGO is designed to extend LIC with a larger chamber space to accommodate regular wells and to serve special requirements, such as complete electrical insulation. In addition, this study cooperates with Academia Sinica to investigate the effect of ultrasound stimulation on the ion channels of DRG neuron cell using the patch-clamp technique. The purpose of this study is to investigate if ultrasound stimulation can open the ion channel on the cell membrane. However, due to its application in electro-physiological experiments, it is necessary to consider the problem of electrical signal interference and chamber space insulation. Another feature of MIGO is the large internal stimulation space, which can directly stimulate the cells in the culture dish stimulating C2C12 cells to observe cell differentiation. As device parameters, the results demonstrate that the system can provide a uniform energy field in the stimulation space, and the intensity (ISATA) in chamber and in culture dish are 1.19 mW/cm2 and 0.84 mW/cm2 respectively. Similarly, the results demonstrated that when the temperature of the incubator is 33oC and four 1 MHz ultrasonic probes were applied at the same time, the temperature in the chamber was stabilized at 36.6 oC in the end.

Probe Adapter: Probe Adapter was designed to solve the problems encountered by MIGO in ultrasonic ion cell ion channel testing experiments. We developed a new set of methods for ultrasound stimulation of cells. It is designed to provide a smooth surface that attaches ultrasonic probe and a hole that connects the stainless steel pipette holding the tip of the glass tube at the front end. The advantage of this setup is that the ultrasonic waves can be transmitted to the glass tip for a localized stimulation. According to the results of the ion channel test for DRG neuron cell by ultrasound, it can be found that the success rate of repeatedly detecting the change of the cell membrane current is improved, thereby improving the repeatability of experiment. In the device parameter measurement, the energy output of the glass tip is about 0.006 to 0.02 mW/cm2 with different versions of Probe Adapter at amplitude of 2Vpp, 3Vpp, 5Vpp, 8Vpp, 10Vpp and 1.1 MHz continuous ultrasound input.

Conclusion: We reported four ultrasound devices in this thesis. LIC, MIGO, and Probe Adapter are self-developed products. The experiments carried out to validate the usage of these devices are live cells imaging under ultrasound stimulation, ultrasound stimulation with culture dish and the ion channel test for DRG neuron cell by ultrasound. From the results of the experiment, it can be concluded that these devices deliver their expected functionality.